On the identification of FOXP2 gene enhancers and their role in brain development Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof.dr. J.H.J.M. van Krieken, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 26 oktober 2016 om 16:30 uur precies door Martin Becker geboren op 21 maart 1985 te Berlijn (Duitsland) Promotor Prof. dr. Simon E. Fisher Copromotor Dr. Sonja C. Vernes (Max Planck Instituut voor Psycholinguistiek) Manuscriptcommissie Prof. dr. Hans H.L.M. van Bokhoven Dr. Annette Schenck Prof. dr. Wolfgang Enard (Ludwig-Maximilians- Universität München, Duitsland) On the identification of FOXP2 gene enhancers and their role in brain development Doctoral Thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof.dr. J.H.J.M. van Krieken, according to the decision of the Council of Deans to be defended in public on Wednesday, October 26, 2016 at 16:30 hours by Martin Becker Born on March 21, 1985 in Berlin (Germany) Supervisor Prof. dr. Simon E. Fisher Co-supervisor Dr. Sonja C. Vernes (Max Planck Institute for Psycholinguistics) Doctoral Thesis Committee Prof. dr. Hans H.L.M. van Bokhoven Dr. Annette Schenck Prof. dr. Wolfgang Enard (Ludwig-Maximilians- Universität München, Germany) "Take responsibility for making your own life beautiful." By Timothy Leary, Your Brain Is God © 2016 Martin Becker All rights reserved. No part of this thesis may be reproduced or printed in any form, by any electronic or mechanical means, without written permission of the author. Cover design: Antje Märtin, Martin Becker Printed by: Ipskamp Drukkers, Enschede Contents Chapter 1: General introduction……………………………………………………………………………………2 Chapter 2: A chromosomal rearrangement in a child with severe speech and language disorder separates FOXP2 from a functional enhancer …………………………………………………….. 31 Chapter 3: Identification of neuronal FOXP2 enhancer contacts in human cell lines……………………….. 38 Chapter 4: Upstream regulatory mechanisms acting at the promoters and enhancers of FOXP2………... 84 Chapter 5: Activity of FOXP2 enhancers during development and in adult brains …………………………. 124 Chapter 6: Early developmental gene enhancers affect subcortical volumes in the adult human brain….. 159 Chapter 7: Summary and General discussion ………………………………………………………………….. 183 Appendix 1….……………………………………………………………………………………………… 207 Appendix 2…………………………………………………………………………………………………. 210 Dutch summary/Nederlandse samenvatting……………………………………………………………. 227 Curriculum Vitae…………………………………………………………………………………………… 231 List of publications…………………………………………………………………………………………. 232 Acknowledgements………………………………………………………………………………………... 233 MPI Series in Psycholinguistics………………………………………………………………………….. 234 1 Chapter 1: General introduction Chapter 1 General introduction 2 Chapter 1: General introduction Introduction The study of genes in health and disease has dramatically increased our understanding of human biology. The identification of mutated genes in diseases of the kidney, liver, muscle or virtually any other human tissue enabled us to study the function of these tissues at molecular and cellular levels. In this dissertation, I describe investigations of the FOXP2 gene, which has been found to be mutated in people with speech and language problems. The relation between FOXP2 and human language was first discovered nearly 15 years ago (Lai et al., 2001) and has been described “as a molecular window into speech and language” (Fisher and Scharff, 2009). My aim was to change angles and look through that window at the molecular mechanisms that precede FOXP2. In this chapter I will introduce FOXP2, starting with the discovery of this gene in a family with a speech and language disorder. I will describe the expression during development and in adult brains and summarize the current knowledge regarding the downstream molecular and cellular functions of FOXP2. Next, I will review the current literature on the upstream processes that may regulate FOXP2 and show that this aspect of the FOXP2 story is not well understood. At the end of this chapter, I will formulate the overarching question and aims of this dissertation. Speech and language problems in people with FOXP2 mutations In 1990, a large three-generation family was described, of which about 50 percent of the family members presented with primary deficits in speech and language (Gopnik, 1990; Hurst et al., 1990) (Figure 1). A major aspect of the disorder is developmental verbal dyspraxia (DVD), also known as childhood apraxia of speech (CAS) (Hurst et al., 1990). The affected members of this family, generally referred to as the KE family, have severe problems with articulation, as well as poor processing and production of grammatical structures. The articulatory problems involve difficulties in performing the rapid coordinated sequences of oral and facial movements required for speech (Hurst et al., 1990; Vargha-Khadem et al., 1995). In addition to the orofacial motor control problems, the affected members showed problems with language, including 3 Chapter 1: General introduction reduced ability in applying grammatical rules governing tenses and plurals (Gopnik and Crago, 1991; Vargha-Khadem et al., 1995) and lower scores on tests for the understanding of grammar (Vargha-Khadem et al., 1995). In terms of general cognition, both unaffected and affected members scored on the lower side of the normal range of the general population. However, a low IQ did not co-segregate with the speech and language disorder (Vargha- Khadem et al., 1995). It has been argued by some that the speech and language problems in this disorder stem from a core deficit affecting the coordination of orofacial motor-movements (Vargha-Khadem et al., 2005). Figure 1: Pedigree of the KE family reproduced from Lai et al. 2001. Affected individuals are indicated by filled symbols. Squares are males, circles are females, and a line through a symbol indicates that the person was deceased in 2001. Analyses of the phenotype of the KE family suggested that disrupted cognitive and neural motor-control functions underlie the observed problems with speech and language development. To identify affected brain regions in the KE family, brain imaging studies were performed on healthy and affected members. In structural magnetic resonance imaging (MRI) studies the grey matter volumes of the affected family members were compared to the unaffected members and an unrelated control group, using voxel based morphometry (Vargha- Khadem et al., 1998; Watkins et al., 2002; Belton et al., 2003). The volumes of the head of the 4 Chapter 1: General introduction caudate nucleus and cerebellar lobule VIIIB were consistently reduced in comparison to control groups, whereas the volume of the putamen was increased. In addition, structural differences were detected in cortical areas involved in motor control and language processing, including increased volumes of the posterior superior temporal gyrus (including Wernicke’s area) and angular gyrus, as well as decreased inferior frontal gyrus (including Broca’s area) and precentral gyrus. The affected brain structures form cortico-striatal and cortico-cerebellar circuits, which are important for planning and exerting motor movements (Middleton and Strick, 2000). In complementary functional brain studies, the caudate nucleus showed increased activity in PET scans performed during word repetition tasks (Vargha-Khadem et al., 1998) and the putamen reduced activity in fMRI scans during semantic language tasks, where the subjects had to generate verbs in combination to heard nouns (Liegeois et al., 2003; Liegeois et al., 2011). In addition, in these studies, under- and over-activation of cortical areas involved in orofacial motor control, such as the precentral gyrus, and language-related cortical areas, such as the inferior frontal gyrus, were detected (Vargha-Khadem et al., 1998; Liegeois et al., 2003; Liegeois et al., 2011). Thus, structural and functional aberrations overlap in the basal ganglia, cerebellum, motor-control area and language-related cortical areas in the affected KE family members. Based on the observed effects in cortico-striatal and cortico-cerebellar networks, it has been suggested that the speech and language phenotype result from problems in planning and performing sequences of orofacial movements (Vargha-Khadem et al., 2005). The mode of inheritance in the KE family suggested that a mutation in a single autosomal gene might account for the speech and language disorder of this family. In 1998, the hypothesis of an autosomal dominant locus was confirmed by molecular studies, and the location of the affected gene was narrowed down to a region on chromosome 7 (Fisher et al., 1998). Using genome-wide linkage analysis in the KE family, the authors identified a region on chromosomal band 7q31 that perfectly segregated with affection status. Further clues to the location of the likely damaged gene came from identification of an unrelated case, referred to as CS (Lai et al., 2000), with a strikingly similar phenotype to that seen in the KE family. This child carried a 5 Chapter 1: General introduction de novo balanced translocation involving reciprocal exchanges between chromosomes 7 and 5, with one breakpoint directly disrupting a newly identified gene on chromosome 7q31, in the region of linkage identified in the KE family. This novel gene, given the name FOXP2, was characterized